Epigenetic inactivation of microRNA in male germ cell cancer Personnel: Lee, Cheung, Chan, Rennert MicroRNAs (miRNAs) is a class of small non-coding RNAs that have been shown to be deregulated in many diseases including cancer. An intertwined connection between epigenetics and miRNAs has been supported by the recent identification of a specific subgroup of miRNAs called "epi-miRNAs" that can directly and indirectly modulate the activity of the epigenetic machinery. Using a genome-wide approach for studying differential methylation in testicular germ cell tumor cell line, we previously identified a novel hypermethylated locus on chromosome 1. Genomic mapping revealed that the hypermethylated region overlapped with a mircroRNA candidate, miR-199a and its upstream promoter region. Bisulfite sequencing and treatment with DNA methyltransferase inhibitor 5-aza in the Ntera-2 testis cancer cells confirmed the same observation in the tiling microarray data. To explore the role of miR-199s, we further examined its methylation and expression patterns in several testicular embryonal carcinoma cell lines (Ntera-2, 833K, NCCIT, Cates-1B and Tera-1), a cultured normal testis cell (HT) and 3 normal testis tissues from health males. Bisulfite sequencing showed that the normal cultured testis cell was unmethylated, while for normal testis tissues it is slightly methylated. Except Cates-1B, all examined testicular embryonal carcinoma cell lines were methylated. Expression of miR-199a, as revealed by real-time qPCR, showed that this miRNA was downregulated in all cancer cell lines as compared to either normal testis cell lines or tissues. A negative correlation between methylation and expression of miR-199a was observed in our study, indicating that DNA methylation is a critical regulator of miR-199a. Downregulation of miR-199a was not restricted in testicular cancer cell lines, but also in primary tissues. We collected total RNA from 9 normal testis biopsy, 8 embryonal carcinomas and 9 se-minomas from patients and analyzed the miR-199a expression by qPCR. After normalization, miR-199a-5p was found to be significantly down-regulated in both embryonal carcinomas and seminomas. Down-regulation in seminomas is more consistent in the analyzed cases, probably due to the more homogeneous nature of this poorly differentiated carcinoma cells. To expand the clinical sample size of testicular cancers, we are analyzing the expression of miR-199a in tissue arrays with LNA-based in situ hybridization. The result from tissue array will help us to understand whether miR-199a is significantly dysregulated in primary tumors. To examine the biological functions of miR-199a, we established stably transfected miR-199a cells (NT2-199a).We investigated the ability of NT2-199a cells to form colonies in soft agar medium. Cells of NT2-199a formed colonies similar to NT2-GFP cells. It was also demonstrated to inhibit cell proliferation. These data suggest that miR-199a may act as an anti-metastasis but not a tumor suppressor miRNA. Bioinformatic analysis on microarray expression data on Ntera-2 cells suggested podocalyxin-like (PODXL) might be one of the targets of miR-199a, which were significantly up-regulated in testicular cancer cell line. Transient transfection of miR-199a into Ntera-2 cells or stable expression of miR-199a suppressed the mRNA and protein level of PODXL. In addition, demethylation treatment of Ntera-2 cells by 5-aza restored expression of miR-199a and decreased PODXL expression. These data support that miR-199a-5p is a negative regulator of PODXL. Sequence analysis revealed two seed sequences of miR-199a in the 3-UTR of PODXL. One of them, denoted as UTR-A, is a highly conserved site in mammals. We cloned the two franking seed sequences into luciferase reporter vector. Co-transfection of luciferase reporter vector with miR-199a-5p significantly suppressed the activity of luciferase that carries the UTR-A site. miR-199a-5p has a minor effect on the non-conserved UTR-B site. Mutation of the seed sequence of UTR-A resulted in loss of suppression function of miR-199a-5p. These data indicate that miR-199a-5p suppresses PODXL through the conserved seed sequence in the 3-UTR.